Traditionally, three different methods have been used for introducing DNA into strains of Bacillus sp. The first method, which is generally useful only for cells of B. subtilis 168, is transformation of competent cells, the second is based on the principles of electroporation, and the third is based on transformation of protoplasts.
Especially the protoplast transformation and the transformation of competent cells are widely used. However, while protoplast transformation functions for a number of different Bacillus sp. it cannot, in general, be accomplished in less than two weeks which makes this method cubersome to use. Furthermore, as mentioned above, transformation of competent cells has generally only been shown to work satisfactory for cells of B. subtilis 168.
It has been shown that some strains of Bacillus may take up DNA by means of conjugation, i.e. by exchange of genetic material mediated by certain "transfer plasmids".
More specifically, Koehler and Thorne (in Journal of Bacteriology, November 1987, pp. 5771-5278) describe a 55 kb plasmid, pLS20, which is capable of mediating transfer of plasmids between Bacillus sp. The transfer of the tetracycline resistance plasmid pBC16 and the Staphylococcus aureus kanamycin resistance plasmid pUB110, respectively, were shown to be mediated by the plasmid pLS20 from a strain of B. subtilis (natto) to strains of the Bacillus spp. B. anthracis, B. cereus, B. licheniformis, B. megaterium, B. pumilus, B. subtilis and B. thuringiensis. Other plasmids were found to be unable to transfer by use of pLS20. The transfer of plasmids mediated by pLS20 was concluded to take place by donation rather than conduction, i.e. without physical association of the two plasmids.
In Journal of Bacteriology, June 1990, pp. 3290-3297 Selinger et al. identify an open reading frame .beta. (ORF-.beta.) region in the nonconjugative plasmids pUB110 and pBC16 and conclude that this region is essential for mobilization of the plasmids by the conjugational plasmid pLS20 or its derivatives. Also another region of pUB110 and pBC16 located 5' to ORF-.beta. (and presumably including oriT) is shown to be necessary for mobilization. ORF-.beta. is acting in trans whereas the other region is cis-acting.
It is an object of the present invention to provide improved systems for introducing DNA into strains of Bacillus sp. and to use these systems for the production of polypeptides of interest.